The present invention relates to tuftsin receptor-specific peptide constructs which are conformationally fixed on complexation with a metal ion. The constructs, which may be peptidomimetic in nature, are useful in pharmaceutical and radiopharmaceutical applications.
Throughout this application, various publications are referred to, each of which is hereby incorporated by reference in its entirety into this application to more fully describe the state of the art to which the invention pertains.
Tuftsin Receptor Peptide Construct. Ser. No. 08/660,697 teaches certain locally restricted peptides, in which the biological-function domain and metal-peptide backbone are combined, and the biological-function domain is specific for the tuftsin receptor found on polymorphonuclear (PMN) granulocytes, monocytes and macrophages.
Native tuftsin is a tetrapeptide of the sequence Thr-Lys-Pro-Arg (SEQ ID NO.1), located as residues 289-292 of the Fc region of the heavy chain of leukokinin (a cytophilic xcex3-globulin). It is liberated by a combination of two cleavages. The C-terminal peptide bond is cleaved in the spleen by splenic enzyme and subsequent cleavage of the N-terminal peptide bond by enzyme leukokininase which occurs on the membranes of the granulocytes where it acts to stimulate phagocytosis. The tuftsin sequence stimulates macrophages and polymorphonuclear granulocytes towards phagocytosis. This sequence thus has a role in the immune system response for fighting infections and bacteria and other invasions. There are specific tuftsin receptors present on granulocytes and macrophages. The receptor density is approximately 50,000-100,000 per cell, with the receptor-tuftsin complex reported to internalize after binding. Thus a peptide specific for the tuftsin receptor may be used in the treatment of certain diseases, as is disclosed generally in U.S. Pat. No. 4,390,528 to V A Najjar and U.S. Pat. No. 5,028,593 to K Nishioka, the teachings of which are incorporated herein by reference.
The ""697 application teaches a precursor peptide, incorporating both a metal ion-binding backbone and a tuftsin receptor-specific biological-function domain, which tuftsin receptor-specific domain is biologically active only on labeling or complexing the metal ion-binding backbone with a metal ion, of the following general formula:
One representative peptide from this series was the sequence Thr-D-Lys-Gly-D-Cys-Arg (SEQ ID NO.2). This peptide displayed very high affinity (KD=1-5 nM) for human leukocytes after its binding to reduced TcO[V]. When complexed to radioactive 99mTcO[V], the peptide localizes to the site of inflammation or infection on i.v. administration. The affinity of the peptide which is not complexed to a metal ion is on the order of KD=10xe2x88x924 M.
The structure of the Thr-D-Lys-Gly-D-Cys-Arg (SEQ ID NO.2) peptide after binding to technetium is as follows: 
The ""697 application teaches that this peptide can similarly be labeled with Re, and that similar peptides can also be designed and synthesized using an N4 metal ion-binding domain, such as Thr-D-Lys-Gly-D-His-Arg (SEQ ID NO.3). Tuftsin receptor-specific peptides disclosed in ""697 include Thr-D-Lys-Gly-D-Cys-Arg (SEQ ID NO.2), Thr-D-Lys-Gly-D-His-Arg (SEQ ID NO.3) and Pro-D-Lys-Gly-D-Cys-Arg (SEQ ID NO.4).
The peptides taught in ""697 may be complexed with a non-radioactive ionic form of rhenium or another suitable isotope, thereby creating a non-radioactive metallopeptide drug for the treatment of disease. Such peptides may also be radiolabeled with a diagnostic metal ion, such as 99mTc, and used to determine sites of concentration of granulocytes and macrophages, such as infections and inflammations, or radiolabeled with a therapeutic metal ion, such as 186Re or 188Re, and used in the treatment of disease.
In addition, tuftin has analgesic and other central nervous system effects. See, e.g., Herman et al., xe2x80x9cCentral Effects of Tuftsin,xe2x80x9d in Antineoplastic, Immunogenic and Other Effects of the Tetrapeptide Tuftsin: a Natural Macrophage Activator, Najjar V A and Freidkin M, eds., New York Academy of Sciences, 1983 [hereinafter Antineoplastic], 156-163; Paradowski et al., xe2x80x9cThe Influence of Tuftsin on Blood Pressure in Animals,xe2x80x9d in Antineoplastic, 164-167; Fridkin and Najjar, Crit. Rev. Biochem. Med. Biol., 24 (1989). Herein disclosed are novel peptides and peptidomimetics which are specific for the tuftsin receptor and may be used as an analgesic and in the treatment of various other central nervous system conditions.
Metallopeptides. The present invention provides tuftsin receptor-specific peptides which comprise a metal ion-binding backbone for complexing with a metal ion, the peptide further comprising a tuftsin receptor-specific biological-function domain, in which the tuftsin receptor-specific domain is conformationally constrained on complexing the metal ion-binding backbone with the metal ion. The metal ion-binding backbone includes two or more contiguous amino acids available for complexing with a metal ion, provided such that the peptide is specific for the tuftsin receptor on complexing the metal ion-binding backbone with a metal ion. The tuftsin receptor-specific domain may be sychnological or rhegnylogical.
The present invention encompasses manufactured peptides and pharmaceutically acceptable salts thereof which are characterized by having a metal ion-binding backbone with two or more contiguous amino acids available for complexing with a metal ion, and a tuftsin receptor-specific biological-function domain which is conformationally constrained on complexing the metal ion-binding backbone with a metal ion. In general, at least a portion of the peptide is conformationally constrained in a secondary structure on complexing the metal ion-binding backbone with the metal ion. The peptide may have a conformationally constrained global structure on complexing the metal ion-binding backbone with the metal ion. The tuftsin receptor-specific domain of the peptide is substantially more potent on complexation of the metal ion-binding backbone with the metal ion. The peptide is also substantially more resistant to enzymatic degradation after complexing the metal ion-binding backbone with a metal ion.
Typically, the metal ion-binding backbone is designed so that all of the valences of the metal ion are satisfied on complexation of the metal ion. In such instances, the metal ion-binding backbone may be a plurality of amino acids each containing at least one nitrogen, sulfur or oxygen atom available for complexing with the available valences of the metal ion. The metal ion-binding backbone also may include a derivatized amino acid or spacer sequence which contains at least one nitrogen, sulfur or oxygen atom available for complexing with the available valences of the metal ion.
The biological-function domain of the tuftsin receptor-specific metallopeptide constitutes a ligand capable of binding with a receptor. The affinity of the tuftsin analog peptide ligand for its receptor will generally be substantially higher when the metal ion-binding backbone is complexed with the metal ion than that of the uncomplexed tuftsin analog ligand.
The metal ion to be complexed may be selected from the group of elements consisting of iron, cobalt, nickel, copper, zinc, manganese, arsenic, selenium, technetium, ruthenium, palladium, silver, cadmium, indium, antimony, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, polonium and astatine. For the peptides of this invention, a metal ion which has a coordination number of 4 and is able to complex with a tetradentate ligand is preferred. The isotope 99mTc is particularly applicable for use in diagnostic imaging, and the isotopes 186Re and 188Re are preferred for therapeutic applications. Non-radioactive rhenium is particularly applicable for use in making non-radioactive metallopeptides.
Tuftsin Analogs. Peptides of this invention may be manufactured peptides and pharmaceutically acceptable salts thereof containing a metal ion-binding backbone including two or more contiguous amino acids available for complexing with a metal ion, and a biological-function domain specific for the tuftsin receptor, which tuftsin receptor-specific domain is conformationally constrained on complexing the metal ion-binding backbone with a metal ion.
The metal ion-binding backbone may be complexed with a gamma-emitting metal ion, and the peptide used for diagnostic imaging of sites of infection or inflammation. The peptide may also be used as an immunostimulatory agent, and may in such instances be complexed with a metal ion which is not radioactive. The foregoing peptides can be complexed with technetium-99m (99mTc) a gamma emitter useful in diagnostic radioimaging, or with either radioactive or non-radioactive isotopes of rhenium.
Accordingly, it is an object of this invention to devise, demonstrate and illustrate the preparation and use of highly specific conformationally restricted peptides, peptoids, related pseudopeptides, peptidomimetics and metallo-constructs formed by complexing sequences thereof to a desired metal ion so that the topography of the side chains in the resulting complex is a biologically active three-dimensional structure which binds to a tuftsin receptor.
Another object of this invention is to provide tuftsin receptor-specific peptide-metal ion complexes which have a higher level of stability and are less susceptible to proteolysis than either the uncomplexed peptide, or other peptides known in the art.
Another object of this invention is to provide for tuftsin receptor-specific peptide analogs which are not conformationally restricted in the absence of a metal ion, whereby the uncomplexed peptide analog is either inactive or demonstrates low potency, but which have high potency and concomitant conformational restriction on complexation with a metal ion.
Another object of this invention is to utilize metal complexation in a tuftsin receptor-specific peptide to cause specific regional conformational restrictions in the peptide so that the peptide conformation at the metal binding site is conformationally fixed on metal complexation.
Another object of this invention is to complex a tuftsin receptor-specific peptide to a metal ion so as to alter the in vivo biodistribution profile, rate and mode of clearance from the body, bioavailability and pharmacokinetics in mammals.
Another object of this invention is to provide tuftsin receptor-specific peptide-metal ion complexes which utilize stable non-radioactive metal ions, with the biological-function domain having specific tuftsin-like biological activity, such as for therapeutic treatment of disease.
Another object of this invention is to provide a molecule which, on complexing with a metal ion, includes a biological-function domain which is specific for tuftsin receptors, and which stimulates polymorphonuclear granulocytes, monocytes and macrophages towards phagocytosis, and may be used in diagnostic methods for abscess and infection imaging.
Another object of this invention is to provide a peptide-metal ion complex with a region specific for the tuftsin receptor on polymorphonuclear granulocytes and macrophages which increases the antigenic profile of antigens presented to such polymorphonuclear granulocytes and macrophages, thereby resulting in production of higher titer antibodies.
Another object of this invention is to develop a tuftsin receptor-specific peptide-metal ion complex which is an antagonist of tuftsin.
Another object of this invention is to develop a tuftsin receptor-specific peptide-metal ion complex which is an agonist of tuftsin.
Another object of this invention is to complex tuftsin receptor-specific peptides with radiometal ions for use in whole body imaging and radiotherapy so that the resulting peptide-metal ion complex is of higher affinity and specificity for the tuftsin receptor than the uncomplexed peptide molecule, and the resulting radiolabeled species is essentially carrier-free in terms of tuftsin receptor recognition.
Another object of this invention is to provide tuftsin receptor-specific peptide-metal ion complexes which can transit the gut-blood barrier, without significant enzymatic or peptidase degradation, and may be adapted for oral administration.
Tuftsin receptor-specific peptide-metal ion complexes, and the precursor uncomplexed sequences, which include peptide, peptidomimetic, peptide-like and metallo-constructs, are provided for biological, pharmaceutical and radiopharmaceutical applications. In the tuftsin receptor-specific peptide-metal ion complexes the construct is conformationally fixed, with the tuftsin receptor-specific domain generally having increased affinity for its target on labeling the metal ion-binding backbone with a metal ion.
The peptide constructs of this invention can include a metal ion, and for embodiments in which the metal ion is used diagnostically or therapeutically, a medically useful metal ion. The metal ion is optionally radioactive, paramagnetic or superparamagnetic. The metal ion is an ionic form of an element selected from the group consisting of iron, cobalt, nickel, copper, zinc, manganese, arsenic, selenium, technetium, ruthenium, palladium, silver, cadmium, indium, antimony, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, polonium and astatine. The metal ion may also be an ionic radionuclide of indium, gold, silver, mercury, technetium, rhenium, tin, astatine or copper.
A radioactive medically useful metal ion may generate gamma rays, beta particles, or positrons which are converted into gamma rays on collision with electrons. The medically useful metal ion may be used in diagnostic imaging procedures including gamma scintigraphy, specific photon emission computerized tomography, or positron emission tomography. The medically useful metal ion may also be used diagnostically in magnetic resonance imaging. Medically useful metal ions may also be used therapeutically.
The type of medically useful metal ion depends on the specific medical application. Particularly useful metal ions include elements 25-30 (Mn, Fe, Co, Ni, Cu, Zn), 33-34 (As, Se), 42-50 (Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn) and 75-85 (Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Po, At). Isotopes of the elements Tc, Re, and Cu are particularly applicable for use in diagnostic imaging and radiotherapy. The isotope 99mTc is particularly applicable for use in diagnostic imaging. Other radionuclides with diagnostic or therapeutic applications include 62Cu, 64Cu, 67Cu, 97Ru, 105Rh, 109Pd, 186Re, 188Re, 198Au, 199Au, 203Pb, 211Pb and 212 Bi.
The tuftsin receptor-specific domain of the peptide is a sequence of one or more amino acids which constitute a biologically active peptide sequence, exhibiting binding to the tuftsin receptor found on cells, tissues or organs. The tuftsin receptor-specific domain also includes any sequence which may be consecutive amino acids (sychnological) or may be non-consecutive amino acids (rhegnylogical), of one or more amino acids which forms a tuftsin receptor-specific ligand, which ligand is capable of forming a specific interaction with its acceptor or receptor. The term xe2x80x9creceptorxe2x80x9d is intended to include both acceptors and receptors. The peptide or the biological-function domain may optionally transmit a signal to the cells, tissues or other materials associated with the biological receptor after binding. The tuftsin receptor-specific domain may thus be either an agonist or antagonist, or a mixed agonist-antagonist. The tuftsin receptor-specific domain may also constitute a member of a xe2x80x9cspecific binding pair,xe2x80x9d wherein a specific binding pair comprises at least two different molecules, where one molecule has an area on the surface or in a cavity which specifically binds to a particular spatial and polar organization of the other molecule.
Radiopharmaceutical Applications. Products of this invention may be employed as radiopharmaceutical agents. For example, when labeled with gamma-emitting radioisotopes, such as 99mTc, the products may be utilized as a diagnostic agent in nuclear medicine.
Products of this invention may also be used as therapeutic agents when labeled with alpha- or beta-emitting radioisotopes. For example, peptides labeled with alpha- or beta-emitting radioisotopes, such as rhenium-186 (186Re) or rhenium-188 (188Re), can be used for treating diseases.
For radiopharmaceutical applications, and other medical applications, the products of this invention offer significant advantages over conventional linear or single-chain peptide constructs. For example, it is known that conformationally constrained and dimeric peptides derived from hypervariable loop sequences of antibodies can bind antigens with an affinity up to 40-fold higher than that obtained with linear sequence peptides. The peptides of this invention are conformationally constrained on labeling with a metal ion, and have a higher affinity than that obtained with conventional linear sequences.
For radiopharmaceutical and other medical applications, the peptides of this invention may be delivered to a subject by any means known in the art. This includes intravenous injection, subcutaneous injection, administration through mucous membranes, oral administration, dermal administration, regional administration to an organ, cavity or region, and the like.
Non-Radiopharmaceutical Therapeutic Applications. The products of this invention may be used for therapeutic applications, and are particularly useful for peptide drugs in which a tuftsin receptor-specific biological-function domain is required. In these applications, the metal ion may serve only to conformationally constrain the peptide, or a portion thereof, or may itself be related to the therapeutic nature of the agent.
Specific Tuftsin Analogs. The peptides of Table 1 were synthesized by solid-phase peptide synthesis using Boc-chemistry, and were purified by HPLC to purity levels of 95% or higher and analyzed by electrospray mass spectrometry. For all products, the experimental and calculated molecular masses were identical.
The peptides of Table 1 are synthesized by any means known in the art, including those methods disclosed in ""697, and are evaluated to determine their ability to complex 99mTc. It was determined that each peptide complexed 99mTc very effectively. Each peptide was labeled using an identical protocol. A 5-10 xcexcg sample of the peptide taken in 0.001 N aq. HCl was mixed with 1-30 mCi of generator-eluted Na99mTcO4 in a 5 ml serum vial. The volume of the resulting mixture was adjusted to 600 xcexcl using injectable saline. A 400 xcexcl volume of a freshly prepared and nitrogen-purged phthalate-tartrate-Sn(II) buffer (40:10:1 mM) was then added to the vial under a nitrogen head space. The vial was immediately sealed and placed in a shielded boiling water bath. After 15 min. the vial was removed from the water bath and allowed to come to room temperature. A small amount of the sample (1-10 mCi) was analyzed by reverse-phase HPLC using a C-18 column (VYDAC, 250xc3x974.8 mm, 10 micron particle size) with a 0-30% acetonitrile gradient in 0.1% aq. TFA completed in 30 min. at a flow rate of 1.5 ml/min. Radioelution profiles were generated using an in-line radioactivity detector (Beckman, Model 170). The Tc-peptide complexes were usually obtained as a mixture characterized by two HPLC peaks, presumptively due to syn- and anti-isomerism in the Tc=O core. The HPLC profiles for each of the 99mTc-peptides showed a complete absence of free, uncomplexed 99mTc (which elutes at 2.5-3 min. under the reverse-phase HPLC conditions described). The radiochemical purity, as calculated from the HPLC profiles, ranged from 90-97%.
The peptides of Table 1 may alternatively be labeled with 99mTc by any of the means taught in ""697, including use of stannous-tartrate-succinate buffer, stannous-EDTA-succinate buffer, stannous stabilized in glucoheptonate, or a stannous-borate-tartrate buffer, as well as other means of labeling with 99mTc known in the art.
The peptides of Table 1 may be complexed with non-radioactive metal ions, and rhenium is a preferred ion. Peptides in solution may be labeled by treatment with the rhenium transfer agent ReO[V]Cl3(PPh3)2 in the presence of 1,8-Diazabicyclo[5,4,0] undec-7-ene as a base. Metal complexation in the presence of 1,8-Diazabicyclo[5,4,0]undec-7-ene as a base can conveniently be accomplished at ambient room temperature. In an alternative method of metal complexation a mild base, such as sodium acetate, can be used. In this case the peptide is taken in a suitable solvent, such as DMF, NMP, MeOH, DCM or a mixture thereof, and heated to 60-70xc2x0 C. with the rhenium transfer agent ReO[V]Cl3(PPh3)2 in the presence of sodium acetate for 15 minutes. Similarly, other bases such as triethylamine, ammonium hydroxide and so on, may be employed. Various mixtures of the solvents, also in combination with MeOH, and DCM, CHCl3 and so on, may also be employed to yield optimized complexation results.
The peptides of Table 1 may be used as diagnostic imaging agents for localizing sites of infection or inflammation, particularly when labeled with 99mTc, or as immunotherapeutic agents, particularly when labeled with radioactive isotopes of rhenium or complexed with non-radioactive isotopes of rhenium, all as described elsewhere herein and in ""697.
The peptides of Table 1 have, on labeling with technetium, 99mTc or a similar metal ion, a core configuration as shown below. In this configuration, each of R1, R2, R3 and R4, if provided, may be one or more amino acids as described herein, or may be other constructs as described herein. Amino acids, if provided, at R2, R3 and R4, may form at least a part of the tuftsin receptor-specific biological-function domain. Amino acids with cationic side chains at two or more of R2, R3 or R4 may form at least a part of the tuftsin receptor-specific biological-function domain. Peptides of this invention with amino acids having cationic side chains at R2, R3 and R4 include: SEQ ID NOS. 19, 21, 22, 23 and 24. Peptides with cationic side chains at R2 and R4 include: SEQ ID NOS. 2, 3, 4, 33, 7, 8, 10, 11, 12, 13, 14, 17, 18, 27, 30 and 32. Peptides with cationic side chains at R3 and R4 include: SEQ ID NOS. 15, 16 and 20.
Peptides as disclosed herein may include either a D- or L-cysteine residue in the metal ion-binding domain and may incorporate a core sequence described by the formula: xe2x80x83R2xe2x80x94R3xe2x80x94Cysxe2x80x94R4
wherein
R2 is D- or L- Lys, Arg, Gly, Thr, Gln or Orn
R3 is D- or L- Gly, Ser, Lys, Arg
Cys is D-Cys or L-Cys and
R4 is D- or L- Arg, Lys, Leu or Orn